The balance of Wnt and FGF signaling influences the rate of neuromast deposition The migrating pLLP contains 2 to 3 proto-neuromasts at progressive stage of maturation. Previous studies have suggested that their formation is induced near the leading end of the pLLP by FGF signaling, which in turn is dependent on FGF ligands, FGF3 and FGF10, secreted in response to Wnt signaling at the leading end of the pLLP. While Wnt signaling drives expression of fgf10 and fgf10 at the leading end of the pLLP, it simultaneously inhibits local FGF signaling by driving expression of sef, which encodes an intracellular inhibitor of FGF signaling. As a consequence, FGF ligands expressed in a leading domain can only effectively activate FGF signaling and initiate proto-neuromast formation in an adjacent trailing domain, where there is less inhibition by Wnt-dependent Sef expression. Cell proliferation adds cells to the leading end of the pLLP and this progressively displaces the newly formed proto-neuromast to a more trailing position, where it continues to mature. Once the leading end has elongated sufficiently, formation of another proto-neuromast is initiated. In this manner the migrating pLLP acquires 2-3 proto-neuromasts at progressive stage of maturation and eventually the most mature proto-neuromast is deposited from the trailing end. Previous studies have emphasized that proliferation is a key determinant of the rate of neuromast deposition as it determines how quickly maturing proto-neuromasts reach a trailing compartment of the pLLP, where expression of the chemokine receptor cxcr7b correlates with the slowing and eventual deposition of cells in the pLLP. However, we had observed that as the pLLP moves from the ear to toward the tip of the tail the size of the Wnt active domain progressively becomes smaller and this correlates with neuromasts being deposited more frequently. This raised the possibility that progressive changes in the balance of Wnt and FGF signaling within the migrating pLLP might also influence the rate of neuromast deposition. To test this hypothesis we used various manipulations to alter the balance of Wnt and FGF signaling in the migrating pLLP and examined how they influence the pattern of neuromast deposition. We used a small molecule inhibitor of Wnt signaling, IWR, to reduce Wnt signaling, and knocked down fgf10 function to reduce FGF signaling system, which we expected would indirectly allow expansion of the Wnt signaling system. We found that inhibition of the Wnt system with IWR accelerated proto-neuromast maturation and it increased the rate of neuromast deposition. On the other hand, knockdown of fgf10 significantly delayed the establishment of a robust trailing FGF signaling system and allowed persistent and expanded Wnt signaling along the entire length of the pLLP for a significant period after its migration had started. This delayed initiation of proto-neuromast formation and neuromast deposition. However, once the FGF system was eventually established by the remaining FGF ligand, FGF3, the Wnt active domain began to shrink, proto-neuromast formation was initiated, and the pLLP began depositing neuromasts. Interestingly, once the Wnt system began to shrink in fgf10 morphant embryos, it shrank faster than in control embryos in which the balance of Wnt and FGF system had not been disturbed. This relatively rapid shrinking of the Wnt active domain correlated with a faster rate of neuromast deposition. Together these observations show that balance of Wnt and FGF signaling is a key determinant of when formation of new proto-neuromasts is initiated, how quickly they mature, and when they are eventually deposited. Previous studies had indicated that the rate of neuromast deposition is determined by the rate of cell proliferation and that Wnt signaling is an important determinant of cell proliferation. However, other studies examining the role of Lef1 in determining Wnt-dependent cell proliferation, suggested that loss of lef1 function results in an accelerated rate of neuromast deposition, just as we had seen following inhibition of Wnt signaling with IWR. To resolve the paradox of why loss of Lef1, which clearly reduced proliferation, was not reducing the rate of neuromast deposition, we hypothesized that loss of Lef1 may both reduce proliferation and have other independent effects that accelerate neuromast formation and deposition. Consistent with this prediction, we went on to demonstrate that while loss of lef1 reduces cell proliferation at the leading end of the pLLP it also specifically reduces expression of Mkp3, an antagonist of FGF signaling, at the leading end of the pLLP. Exposure of the embryos to a small molecule inhibitor of Mkp3 function resulted in similar accelerated neuromast deposition. These observations suggest that loss of Lef1-dependent expression of this specific inhibitor of FGF signaling allows more rapid establishment of a robust FGF signaling system and, during the course of pLLP migration, more rapid shrinking of the Wnt active domain at the leading end of the pLLP. This contributes to accelerated neuromast formation and deposition. We were also able to show that another component of the Wnt signaling pathway, R-spondin, whose expression is also dependent on Wnt signaling, is essential for Wnt-dependent cell proliferation. As it is a component of the Wnt signaling pathway that primarily determines cell proliferation, loss of R-Spondin function, does not accelerate neuromast deposition, instead, as predicted by studies that have shown cell proliferation promotes neuromast deposition, it results in less frequent neuromast deposition.